1. Field of the Invention
The present invention relates to a fluorescence microscope, and more particularly, to a fluorescence microscope including a wavelength selection filter inclined with respect to light.
2. Description of the Related Art
As a general configuration of a fluorescence microscope, there is known a configuration in which a dichroic mirror is inclined at 45 degrees with respect to the optical axis of incident light (excitation light and fluorescence) at a position where an illumination optical path and an observation optical path intersect. In the configuration, the dichroic mirror can reflect the excitation light toward a sample, and can also transmit the fluorescence emitted from the sample and guide the fluorescence to a detector since the fluorescence has a different wavelength from that of the excitation light projected onto the sample.
FIGS. 1A and 1B are views illustrating the wavelength characteristics of a dichroic mirror inclined with respect to light. The dichroic mirror functioning as a wavelength selection filter is normally formed of interference films. Thus, when light enters the dichroic mirror at an inclined angle, wavelength characteristics with respect to P-polarized light and S-polarized light differ from each other, and a reflection band for the S-polarized light is wider than a reflection band for the P-polarized light in the wavelength characteristics as illustrated in FIGS. 1A and 1B.
When the fluorescence microscope is a laser microscope, a laser beam as excitation light is emitted as linearly-polarized light. Meanwhile, fluorescence emitted from a sample is randomly-polarized light. Excitation light reflected from the sample or the like (referred to as return light below so as to be distinguished from the excitation light that travels from a light source toward the sample) is also randomly-polarized light due to the influence of scattering at a sample surface or the like.
To ensure a widest transmission band for the fluorescence as the randomly-polarized light, the characteristic with a wider reflection band with respect to the S-polarized light is preferably designed according to the excitation wavelength of the laser beam, to allow the laser beam to enter the dichroic mirror as the S-polarized light. FIG. 1A illustrates the wavelength characteristics of the dichroic mirror designed according to the laser beam as the S-polarized light.
In the wavelength characteristics illustrated in FIG. 1A, the reflection band for the S-polarized light is limited to a narrow band including the excitation wavelength. Thus, high transmittance is achieved for the fluorescence wavelength. On the other hand, the reflection band for the P-polarized light is narrower than the band of the excitation wavelength. Thus, a P-polarized component of the return light is partially transmitted through the dichroic mirror together with the fluorescence. Therefore, the fluorescence and the return light cannot be completely separated only by using the dichroic mirror having the wavelength characteristics illustrated in FIG. 1A.
To block the return light by reflecting the return light at the dichroic mirror, the characteristic with respect to the P-polarized light, which is a narrower reflection band than that with respect to the P-polarized light, is preferably designed according to the excitation wavelength of the laser beam. FIG. 1B illustrates the wavelength characteristics of the dichroic mirror designed according to the laser beam as the P-polarized light.
In the wavelength characteristics illustrated in FIG. 1B, both the reflection band for the S-polarized light and the reflection band for the P-polarized light include the excitation wavelength. Thus, the dichroic mirror can reflect the excitation light toward the sample, and can also block the return light. Meanwhile, the reflection band for the S-polarized light is formed wider than that in the wavelength characteristics illustrated in FIG. 1A. Thus, a portion of the band of the fluorescence wavelength is included in the reflection band for the S-polarized light. An S-polarized component of the fluorescence is thereby partially reflected by the dichroic mirror and blocked together with the return light. Therefore, when the dichroic mirror having the wavelength characteristics illustrated in FIG. 1B is used, the detection efficiency of the entire apparatus for the fluorescence is lowered.
As described above, in the dichroic mirror inclined with respect to the light, it is difficult to achieve high transmittance for the fluorescence wavelength and also block the return light due to a difference between the wavelength characteristics with respect to the S-polarized light and the P-polarized light.
To solve the problem, as a general configuration of the fluorescence microscope, a barrier filter that blocks the return light is arranged on the detector side of the dichroic mirror along with the dichroic mirror having the wavelength characteristics illustrated in FIG. 1A for achieving high transmittance for the fluorescence wavelength. With the configuration, high detection efficiency can be achieved for the fluorescence, and the return light can be also blocked.
Japanese Patent Laid-Open No. 2008-33263 discloses the configuration of a fluorescence-testing scanning laser microscope including a beam splitter that is arranged such that the incident angle of illumination light and/or sample light at a splitter surface is smaller than 45 degrees. Generally, as the incident angle is smaller, the difference between the wavelength characteristics with respect to the S-polarized light and the P-polarized light is decreased. With the configuration disclosed in Japanese Patent Laid-Open No. 2008-33263, high detection efficiency can be achieved for the fluorescence, and the return light can be also more reliably blocked by arranging the beam splitter in such a manner as to reduce the incident angle.